Bridges Lab Protocols - User contributions [en]

BDNF PCR

2014-04-01T14:13:45Z

Mpeloqu1: Created page with "'''BDNF Primers''' ---- * oIMR0133 ---- ATG AAA GAA GTA AAC GTC CAC (Common) * oIMR0113 ---- CCA GCA GAA AGA GTA GAG GAG (WT Reverse) * oIMR01694 --- GGG AAC TTC CTG ACT AG..."

'''BDNF Primers'''

----
* oIMR0133 ---- ATG AAA GAA GTA AAC GTC CAC (Common)

* oIMR0113 ---- CCA GCA GAA AGA GTA GAG GAG (WT Reverse)

* oIMR01694 --- GGG AAC TTC CTG ACT AGG GG (Reverse)

'''BDNF Cycler Program'''
----
#94C - 10 minutes (HotStart Taq)
#94C - 30 seconds
#55C - 30 seconds
#72C - 30 seconds ----- Repeat steps 2-4 for 35 cycles
#72C - 2 minutes
#4C - Hold
----
http://jaxmice.jax.org/protocolsdb/f?p=116:2:3011685288607993::NO:2:P2_MASTER_PROTOCOL_ID,P2_JRS_CODE:339,002266

Main Page

2014-04-01T14:03:54Z

Mpeloqu1: Added BNDF

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]
*[[Designing and Generating CRISPR-Cas Mutants]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]
*[[BDNF PCR]]
===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]
*[[Paraffin Embedding of Tissue Samples]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Rapamycin Injections

2014-02-19T15:03:00Z

Mpeloqu1:

==Materials==
*Tween 80 - Sigma P1754
*PEG 400 Liquid - Sigma P3265

==Preparation==
*Weigh out 2.08g of each Tween 80 and PEG into a 50 mL falcon tube. Bring up to 40 mL with water and filter by steriflip. This is 5.2% PEG/Tween
*Dissolve rapamycin at 20 mg/mL (0.0219 M) in ethanol (prepare as a 1 mL aliquot and store at -20 C).
*Add 5 uL of 20 mg/mL RAPA in 1 mL Tween 80/PEG diluent to get a 0.1 mg/mL concentration (109 uM; use this for 1 week).
*IP inject 10 uL/g body weight (1 mg/kg)

==Alternate Rapamycin Injection Regimen==
* Phenotypes seem to be different with rapamycin as treated by Lamming et al.
* This protocol is from Lamming et al http://dx.doi.org/10.1126/science.1215135
<blockquote>
rapamycin treatment was performed by injecting 8 to 10 week old mice intraperitoneally
once daily with either 1 mg/kg rapamycin suspended in 0.9% NaCl and 2% ethanol at a
concentration of 0.5 mg/mL (547 μM), or vehicle only for 14-28 days.
</blockquote>

Paraffin Embedding of Tissue Samples

2013-11-01T15:09:46Z

Mpeloqu1: Created page with " == Protocol == #After harvesting tissue from animal, place into pathology cassette and fix in 4% formaldehyde, 4% paraformaldehyde or 4% formalin O/N #After fixation, begin d..."

== Protocol ==
#After harvesting tissue from animal, place into pathology cassette and fix in 4% formaldehyde, 4% paraformaldehyde or 4% formalin O/N
#After fixation, begin dehydrating the tissue in 70% Ethanol
#On the day of planned embedding, wash sample with 100% Ethanol 3 times for 30 minutes.
##While washing in 100% Ethanol, start the paraffin wax machine (Link Building, Room 311)
#After 100% Ethanol washes, Wash 3 times in Xylenes
#Wash samples in a beaker filled with wax at 58-60C, 3 times for 15 mins
#Immediately before embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with wax
#Take your sample and orient it on top of the base wax as desired
#Place pathology cassette on top of the mold and fill with wax covering the mold
#Place mold with sample on "Cold Side" of embedding station
##Wax will solidify in 10-15 minutes
#Remove wax embedded sample from the mold (should just slip out)
#Store at room temperature until prepped to section

Main Page

2013-11-01T14:50:15Z

Mpeloqu1: /* Tissue Preparation */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]

===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]
*[[Paraffin Embedding of Tissue Samples]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Fly Food Protocol

2013-09-13T13:56:29Z

Mpeloqu1: /* Cooking Insturctions */

== Quantities ==

'''Summer'''
#900mL - Corn Syrup
#202.4g - Yeast
#117.2g - Soy Flour
#856g - Corn Meal
#67.6g - Agar
#15L - Water
#65mL - Propionic Acid
#13mL - Green Food Coloring

'''Winter'''
#900mL - Corn Syrup
#202.4g - Yeast
#117.2g - Soy Flour
#856g - Corn Meal
#67.6g - Agar
#17.5L - Water
#65mL - Propionic Acid
#13mL - Green Food Coloring

== '''Cooking Instructions''' ==

#Fill 10-12 empty trays with empty vials
#Measure out correct amount of ingredients using scale in cabinet
#Assemble the mixing apparatus
#Pour '''10 Liters''' of '''Water''' into the pot and start the mixer
#Add '''900mL''' of '''Corn Syrup (Karo)''' into the pot and allow it to mix
#Turn on the steamline, slowly at first but progressively more as the pot begins to heat up
#Once temperature has reached '''60C''' (use thermometer), add in '''67.6g''' of '''Agar'''
##Allow Agar to mix fully into solution. If Agar does not go into solution, the food is unusable
#After Agar has gone into solution, add '''856g''' of '''Corn Meal'''
##Allow Corn Meal to mix well to reduce clumps in food
#Slowly add '''202.4g''' of '''Yeast''' and '''117.2g''' of '''Soy Flour'''
##Some clumps and chunks will be present, however should go away through mixing
#Bring temperature of food above '''80C''' (edges of pot should be boiling)
#Add '''2.5 Liters''' of '''Water''' to food
#Bring temperature of food above '''80C''' (edges of pot should be boiling)
#Add final '''2.5 Liters''' of '''Water''' to food
#Bring temperature of food above '''80C'''
#Completely turn off steamline
#In a 250mL flask, add '''65mL Propionic Acid''' and '''13mL Green Food Coloring'''
##Mix Propionic Acid and Food Coloring thoroughly by swirling
#Once food temperature has fallen below '''80C''', pour in the Propionic Acid/Food Coloring mixture (will turn food green)
#Pour food into 5 Liter pitcher and use vial filling apparatus
#Fill vials ~1 Inch full
#Make sure to thoroughly clean all equipment (mixers, pot, vial apparatus, pitchers)

Fly Food Protocol

2013-09-03T17:37:27Z

Mpeloqu1: /* Cooking Insturctions */

== Quantities ==

'''Summer'''
#900mL - Corn Syrup
#202.4g - Yeast
#117.2g - Soy Flour
#856g - Corn Meal
#67.6g - Agar
#15L - Water
#65mL - Propionic Acid
#13mL - Green Food Coloring

'''Winter'''
#900mL - Corn Syrup
#202.4g - Yeast
#117.2g - Soy Flour
#856g - Corn Meal
#67.6g - Agar
#17.5L - Water
#65mL - Propionic Acid
#13mL - Green Food Coloring

== '''Cooking Insturctions''' ==

#Fill 10-12 empty trays with empty vials
#Measure out correct amount of ingredients using scale in cabinet
#Assemble the mixing apparatus
#Pour '''10 Liters''' of '''Water''' into the pot and start the mixer
#Add '''900mL''' of '''Corn Syrup (Karo)''' into the pot and allow it to mix
#Turn on the steamline, slowly at first but progressively more as the pot begins to heat up
#Once temperature has reached '''60C''' (use thermometer), add in '''67.6g''' of '''Agar'''
##Allow Agar to mix fully into solution. If Agar does not go into solution, the food is unusable
#After Agar has gone into solution, add '''856g''' of '''Corn Meal'''
##Allow Corn Meal to mix well to reduce clumps in food
#Slowly add '''202.4g''' of '''Yeast''' and '''117.2g''' of '''Soy Flour'''
##Some clumps and chunks will be present, however should go away through mixing
#Bring temperature of food above '''80C''' (edges of pot should be boiling)
#Add '''2.5 Liters''' of '''Water''' to food
#Bring temperature of food above '''80C''' (edges of pot should be boiling)
#Add final '''2.5 Liters''' of '''Water''' to food
#Bring temperature of food above '''80C'''
#Completely turn off steamline
#In a 250mL flask, add '''65mL Propionic Acid''' and '''13mL Green Food Coloring'''
##Mix Propionic Acid and Food Coloring thoroughly by swirling
#Once food temperature has fallen below '''80C''', pour in the Propionic Acid/Food Coloring mixture (will turn food green)
#Pour food into 5 Liter pitcher and use vial filling apparatus
#Fill vials ~1 Inch full
#Make sure to thoroughly clean all equipment (mixers, pot, vial apparatus, pitchers)

Fly Food Protocol

2013-09-03T17:36:14Z

Mpeloqu1: Created page with " == Quantities == '''Summer''' #900mL - Corn Syrup #202.4g - Yeast #117.2g - Soy Flour #856g - Corn Meal #67.6g - Agar #15L - Water #65mL - Propionic Acid..."

== Quantities ==

'''Summer'''
#900mL - Corn Syrup
#202.4g - Yeast
#117.2g - Soy Flour
#856g - Corn Meal
#67.6g - Agar
#15L - Water
#65mL - Propionic Acid
#13mL - Green Food Coloring

'''Winter'''
#900mL - Corn Syrup
#202.4g - Yeast
#117.2g - Soy Flour
#856g - Corn Meal
#67.6g - Agar
#17.5L - Water
#65mL - Propionic Acid
#13mL - Green Food Coloring

== '''Cooking Insturctions''' ==

#Fill 10-12 empty trays with empty vials
#Measure out correct amount of ingredients using scale in cabinet
#Assemble the mixing apparatus
#Pour '''10 Liters''' of '''Water''' into the pot and start the mixer
#Add '''900mL''' of '''Corn Syrup (Karo)''' into the pot and allow it to mix
#Turn on the steamline, slowly at first but progressively more as the pot begins to heat up
#Once temperature has reached '''60C''' (use thermometer), add in '''67.6g''' of '''Agar'''
##Allow Agar to mix fully into solution. If Agar does not go into solution, the food is unusable
#After Agar has gone into solution, add '''856g''' of '''Corn Meal'''
##Allow Corn Meal to mix well to reduce clumps in food
#Slowly add '''202.4g''' of '''Yeast''' and '''117.2g''' of '''Soy Flour'''
##Some clumps and chunks will be present, however should go away through mixing
#Bring temperature of food above '''80C''' (edges of pot should be boiling)
#Add '''2.5 Liters''' of '''Water''' to food
#Bring temperature of food above '''80C''' (edges of pot should be boiling)
#Add final '''2.5 Liters"'' of '''Water'''
#Bring temperature of food above '''80C'''
#Completely turn off steamline
#In a 250mL flask, add '''65mL Propionic Acid''' and '''13mL Green Food Coloring'''
##Mix Propionic Acid and Food Coloring thoroughly by swirling
#Once food temperature has fallen below '''80C''', pour in the Propionic Acid/Food Coloring mixture (will turn food green)
#Pour food into 5 Liter pitcher and use vial filling apparatus
#Fill vials ~1 Inch full
#Make sure to thoroughly clean all equipment (mixers, pot, vial apparatus, pitchers)

Main Page

2013-09-03T16:29:49Z

Mpeloqu1: /* Fly Stocks */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]

===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

RIPA Buffer

2013-08-07T15:23:19Z

Mpeloqu1: /* RIPA Buffer (for 10mL lysis buffer) */

==RIPA Buffer (for 10mL lysis buffer)==
{| border="1"
! || Final Concentration || per 10 mL || Stock || Location
|-
|Tris pH7.4 || 50mM || 500uL || 1M || Chemical Shelf
|-
|Na Deoxycholate || 0.25% || 250uL || 10% || Chemical Shelf
|-
| NP-40 || 1% || 1mL || 10% || Chemical Shelf
|-
|NaCl || 150mM || 375uL || 4M || Chemical Shelf
|-
|EDTA || 1mM || 20uL || 0.5M ||Chemical Shelf
|-
|NaVO3 || 100uM || 10uL || 100mM || Chemical Shelf
|-
|NaF || 5mM || 100uL || 500mM || Chemical Shelf
|-
|NaPPi || 10 mM || 1 mL || 100mM || Chemical Shelf
|-
| Protease Inhibitors || || 1 mini tablet || || 4C
|}
[[ Category:Buffer ]]

RIPA Buffer

2013-08-07T15:22:40Z

Mpeloqu1: /* RIPA Buffer (for 10mL lysis buffer) */

==RIPA Buffer (for 10mL lysis buffer)==
{| border="1"
! || Final Concentration || per 10 mL || Stock || Location
|-
|Tris pH7.4 || 50mM || 500uL || 1M || Chemical Shelf
|-
|Na Deoxycholate || 0.25% || 250uL || 10% || Chemical Shelf
|-
| NP-40 || 1% || 1mL || 10% || Chemical Shelf
|-
|NaCl || 150mM || 375uL || 4M || Shelf
|-
|EDTA || 1mM || 20uL || 0.5M || Shelf
|-
|NaVO3 || 100uM || 10uL || 100mM || Chemical Shelf
|-
|NaF || 5mM || 100uL || 500mM || Chemical Shelf
|-
|NaPPi || 10 mM || 1 mL || 100mM || Chemical Shelf
|-
| Protease Inhibitors || || 1 mini tablet || || 4C
|}
[[ Category:Buffer ]]

RIPA Buffer

2013-08-07T15:21:53Z

Mpeloqu1: /* RIPA Buffer (for 10mL lysis buffer) */

==RIPA Buffer (for 10mL lysis buffer)==
{| border="1"
! || Final Concentration || per 10 mL || Stock || Location
|-
|Tris pH7.4 || 50mM || 500uL || 1M || Shelf
|-
|Na Deoxycholate || 0.25% || 250uL || 10% || Shelf
|-
| NP-40 || 1% || 1mL || 10% || Tingting
|-
|NaCl || 150mM || 375uL || 4M || Shelf
|-
|EDTA || 1mM || 20uL || 0.5M || Shelf
|-
|NaVO3 || 100uM || 10uL || 100mM || Biochemical Stocks (-20)
|-
|NaF || 5mM || 100uL || 500mM || Shelf
|-
|NaPPi || 10 mM || 1 mL || 100mM || Shelf
|-
| Protease Inhibitors || || 1 mini tablet || || 4C
|}
[[ Category:Buffer ]]

Maintenance of 18C Fly Stocks

2013-08-06T16:09:48Z

Mpeloqu1: /* =Maintenance */

==Location==
Drosophila stock vials can be found in the 18C cooler of the Reiter Laboratory

==Maintenance==
*Drosophila stocks are to be flipped every 3 weeks
*When flipping the stocks, always flip the oldest of the 2 stock vials
*Once flies have been flipped into new media, label and date the vial
*Dispose of old vial into biohazard waste container
*Place drosophila stocks back into 18C cooler

Maintenance of 18C Fly Stocks

2013-08-06T16:09:20Z

Mpeloqu1: Created page with "==Location== Drosophila stock vials can be found in the 18C cooler of the Reiter Laboratory ==Maintenance= *Drosophila stocks are to be flipped every 3 weeks *When flipping t..."

==Location==
Drosophila stock vials can be found in the 18C cooler of the Reiter Laboratory

==Maintenance=
*Drosophila stocks are to be flipped every 3 weeks
*When flipping the stocks, always flip the oldest of the 2 stock vials
*Once flies have been flipped into new media, label and date the vial
*Dispose of old vial into biohazard waste container
*Place drosophila stocks back into 18C cooler

Main Page

2013-08-06T16:04:13Z

Mpeloqu1: /* Fly Stocks */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]

===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Maintanence of 18C Fly Stocks

2013-08-06T16:02:28Z

Mpeloqu1: Created page with "==Location== Drosophila stocks can be found in the 18C cooler of the Reiter Lab ==Maintenance== *Drosophila stocks are to be "flipped" every 3 weeks *Always flip the oldest o..."

==Location==
Drosophila stocks can be found in the 18C cooler of the Reiter Lab

==Maintenance==
*Drosophila stocks are to be "flipped" every 3 weeks
*Always flip the oldest of the 2 stock vials
*After flipping flies into a new vial, throw away the old vial into the red biohazard container
*Date and label the new vial
*Place stocks back into the 18C cooler

Main Page

2013-08-06T15:55:53Z

Mpeloqu1: /* Mouse/Fly Protocols */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
==Fly Stocks==
*[[Maintanence of 18C Fly Stocks]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]

===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Main Page

2013-08-06T15:54:35Z

Mpeloqu1: /* Mouse Protocols */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]

===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Triglyceride Assay from Drosophila (German Method)

2013-08-01T14:29:47Z

Mpeloqu1: /* Protocol */

==Protocol==
#Use 5 female or 8 Male flies for Assay
#Homogenize flies (3 min @ 40Hz) in 1mL of .05% Tween (from Tween-20 stock)
#Heat samples in 70C waterbath for 5 minutes
#Spin samples @ 5000G for 1 minute
#Transfer 500ul of supernatent in new microfuge tube
#Spin @ 14000G for 3 minutes
#Add 50ul of sample to 200ul of TG solution in a 96 well plate
#Incubate plate @ 37C for 5 mins
#Measure 540nm absorbance

==Publication of Method==
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0023796

Triglyceride Assay from Cells and Tissues

2013-07-31T21:26:30Z

Mpeloqu1: /* Protocol */

==Materials==
* '''Homogenization Buffer''' (50 mM Tris pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
* 10M KOH
* '''Chloroform/Methanol Mixture''' (2:1)
* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
* Sigma Triglyceride Assay Kit (Cat TR0100)

==Protocol==
#Weigh out 30mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
#Add 500ul Homogenization Buffer
#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
#Add 12.5ul KOH
#Mix by inverting
#Add 800ul '''Chloroform/Methanol Mixture'''
#Vortex vigorously then sit at room temperature for 5 minutes
#Centrifuge for 10 minutes @ 13000G
#Transfer the bottom layer into a new tube
#Let evaporate overnight at room temperature
#If absorbance is going to be measured by cuvette, use non-bolded values. If using a plate reader, used bolded values.
#Add 500ul '''(50ul)''' of '''Butanol Mixture'''. See Suggested Volumes for your specific tissue.
#Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
##Resuspend triglyceride and glycerol reagent with water if necessary
##Calculate how many sample you have (samples + blank + standard curve)
##Prepare reagent. You need 560ul '''(80ul)''' of glycerol reagent and 140ul '''(20ul)''' of triglyceride reagent. Make extra and combine in a Falcon tube.
##Aliquot 700ul into a cuvette or '''100ul into a well of a 96 well plate'''
##For standars, add 0-5ul of glycerol standard
##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
##Let sit for ~30 mins @ room temperature (or 5mins @ 37C if you are in a hurry). If using >10ul of butanol mix the solution may be cloudy. Let it settle and it should become more clear.
##Measure absorbance @ 540nm
##If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.

Triglyceride Assay from Drosophila (German Method)

2013-07-31T21:12:37Z

Mpeloqu1: Created page with "==Protocol== #Use 5 female or 8 Male flies for Assay #Homogenize flies (3 min @ 40Hz) in 1mL of .05% Tween (from Tween-20 stock) #Heat samples in 70C waterbath for 5 minutes #..."

Triglyceride Assay from Cells and Tissues

2013-07-31T21:11:21Z

Mpeloqu1: /* Protocol */ Creation of Protocol

Main Page

2013-07-31T21:05:23Z

Mpeloqu1: /* Protein Purification */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse Protocols==
===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]

===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

Preparation of Protein Lysates from Mouse Tissues

2013-07-26T21:27:00Z

Mpeloqu1: /* Protocol */

==Materials==
*RIPA Buffer (see [[Buffer/RIPA|RIPA]]) or other Lysis buffer. Add protease inhibitors.
*Mouse Tissues (Frozen)

==Protocol==
#Cut frozen tissue on a glass plate on dry ice. Place in a new round bottom eppendorf tube.
#Weigh frozen tissue samples, only need 20-50 mg of tissue. If there is too much cut it off and return the extra tissue to the -80. Record the weight of each tissue.
#Add 20 uL/mg of RIPA or other buffer to tissue (400-1000 uL)
#Add a stainless steel bead and keep tissues on ice. Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and 5 min at 50 Hz for Muscle).
#Centrifuge at 14 000 RPM at 4C for 10 min
#Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
#Follow Protein Lysate instructions for Bradford Assay (see [[Bradford Assay]])
#Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of 2X loading buffer with B-ME to each lyaste. This will generate a 2 mg/mL protein solution in SDS Loading Buffer
#Heat samples with loading buffer at 95C for 5 mins
#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80

[[Category:Protein]]
[[Category:SDS-PAGE]]
[[Category:Mouse Work]]
[[Category:Tissues]]

Bradford Assay

2013-07-26T21:23:58Z

Mpeloqu1: /* Protocol */ Added Protein Lysate

==Materials==
*BioRad Protein Assay Dye Reagent Concentrate cat#500-0006
*Disposable Plastic Cuvette

==Protocol==
Cuvette Bradford Assay
#Dilute reagent 5X in water, stable for 2-3 weeks
#Pipet 1 mL into disposable plastic cuvette
#Add 1-10 uL of protein sample, cover with parafilm and mix
#Let sit 5-10 min to react
#Set spectrophotometer as follows:
##Go to protein assay then Bradford assay
##Set formula, then select more
##Set b=0.045 (or determine slope)
##Set dilution to be 1/vol (ie 0.1 for 10 uL)
##Blank then measure samples, absorbance must be less than 0.9
##Print (hit Recall, then enter, then print) and attach to experiment

Protein Lysate Bradford Assay
#Dilute reagent 5X in water, stable for 2-3 weeks
#In a 96 well plate, dilute sample 20X (190ul Reagent, 10ul Sample)
#Add 5ul of sample to 100ul of reagent in well
#Run "Bradford Assay Protocol" on Plate Reader

==Reference==
*Wikipedia: [[wikipedia:Bradford_protein_assay|Bradford Protein Assay]]
*PMID 942051

PCR Analysis of Tail DNA

2013-07-26T13:55:52Z

Mpeloqu1: /* Protocol */

see [[Genotyping Details]] for strain specific details

==Materials==

==Protocol==
Use the following Volumes per 50ul Reaction:

#10X GoTaq Buffer: 5uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 5ul
#dNTPs: 0.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
#Sterile water: 29ul
#Polymerase Go-Taq: 0.125uL ("Molecular Biology Stuff" box in freezer)
#Template: 1 uL

Master Mix (Per 5mL -- Make 1mL Aliquots)
#10X GoTaq Buffer: 625uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 625ul
#dNTPs: 62.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
#Sterile water: 3625ul
#Polymerase Go-Taq: 15.625ul ("Molecular Biology Stuff" box in freezer)
*Add Template Individually

Run PCR Program (approx 2 hours).
Use Cycler 1 on 6th Floor
*Login: Sergey, Just press enter to Login
*Under Genotype folder, pick Ingles program for Ingles genotyping
*Under Genotype folder, pick regpcr program for PLT genotyping

Make sure to press enter 2x once to confirm Tubes and second time to start PCR

see [[Preparing an Agarose Gel]] for details on preparing a DNA gel

[[Category: Genotyping]]
[[Category: Mouse Work]]

PCR Analysis of Tail DNA

2013-07-25T15:00:10Z

Mpeloqu1: /* Protocol */ Master Mix

see [[Genotyping Details]] for strain specific details

==Materials==

==Protocol==
Use the following Volumes per 50ul Reaction:

#10X GoTaq Buffer: 5uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 5ul
#dNTPs: 0.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
#Sterile water: 29ul
#Polymerase Go-Taq: 0.125uL ("Molecular Biology Stuff" box in freezer)
#Template: 1 uL

Master Mix
#10X GoTaq Buffer: 50uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 50ul
#dNTPs: 5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
#Sterile water: 290ul
#Polymerase Go-Taq: 1.25ul ("Molecular Biology Stuff" box in freezer)
*Add Template Individually

Run PCR Program (approx 2 hours).
Use Cycler 1 on 6th Floor
*Login: Sergey, Just press enter to Login
*Under Genotype folder, pick Ingles program for Ingles genotyping
*Under Genotype folder, pick regpcr program for PLT genotyping

Make sure to press enter 2x once to confirm Tubes and second time to start PCR

see [[Preparing an Agarose Gel]] for details on preparing a DNA gel

[[Category: Genotyping]]
[[Category: Mouse Work]]

Western Blotting

2013-07-11T19:39:22Z

Mpeloqu1: /* Protocol */ LiCor Addition

==Materials==
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
*Transfer Apparatus, either Bio-Rad or Invitrogen

==Protocol==
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
#Make sandwich, ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (on the bench).
#Carefully remove blot, stain with Ponceau solution and rinse with TTBS until all the red is washed off
#Block with 2% BSA in [[ TBST ]] or 5% skim milk powder in TBST for >1h
#Incubate with primary antibody (check for dilution) in 1 mg/mL BSA for >1h
#Wash blot every 5 minutes for 15 min with TBST.
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h
#Wash blot every 5 minutes for 15 min with TBST.
#Rinse once or twice with double distilled water
#Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution).
#Drain excess buffer from blot and cover with ECL for about a minute
#Drain excess ECL from blot, cover with saran wrap and expose film
==If Using LiCor==
#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan

[[ Category: Western Blotting ]]

Transfer Buffer

2013-07-11T19:34:41Z

Mpeloqu1: /* 10X Transfer Buffer */ Changed from 10L total to 4L total

==10X Transfer Buffer==
To make 1X TBS, dilute 10X with water, and add 20% methanol. For example:
* 100 mL 10X Buffer
* 200 mL Methanol
* 700 mL Water

{| border="1"
! Component || Weight per 4 L || Catalog Number
|-
|Tris Base || 121.2 g || Sigma T6066-5KG
|-
|Glycine || 576.4 g || Sigma G7126-5KG
|}
[[ Category:Buffer ]]
[[ Category:Western Blotting ]]

Western Blotting

2013-07-11T19:33:23Z

Mpeloqu1: /* Protocol */ Changed wash times

Triglyceride Assay from Cells and Tissues

2013-06-28T17:10:19Z

Mpeloqu1: /* Protocol */ Volume of butanol mixture

==Materials==
* '''Homogenization Buffer''' (50 mM Tris pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
* 10M KOH
* '''Chloroform/Methanol Mixture''' (2:1)
* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
* Sigma Triglyceride Assay Kit (Cat TR0100)

==Protocol==
# Weigh out 30 mg tissue (record weight for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
# Add 500 uL Homogenization Buffer.
# Homogenize with qiagen tissue lyser 3 minutes at 25 Hz for Liver/WAT or 5 min at 30 Hz for muscle.
# Add 12.5 uL KOH
# Mix by inverting
# Add 800 uL '''Chloroform/Methanol Mixture'''
# Vortex vigorously then sit at room temperature for 5 min
# Centrifuge 10min at 13 000 RPM
# Take the bottom layer into a fresh labelled tube.
# Dry in fume hood overnight (or until completely dry)
# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
# Add 500uL '''(50uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
## Resuspend triglyceride and glycerol reagent with water if necessary.
## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
## Prepare reagent, you need 560 uL '''(80uL)''' of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent. Make a bit extra and combine in a falcon tube.
## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
## For standards add 0-5 uL of glycerol standard.
## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
## Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry). If using > 10 uL of butanol mixture the solution may be cloudy, let it settle and it should become more clear.
## Measure absorbance at 540 nm.
## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.

Preparation of Tail Samples (for Genotyping)

2013-06-26T17:18:14Z

Mpeloqu1: /* Protocol */ Proteinase K

==PBND Solution: Tail Lysis Buffer==
* 50mM KCl
* 10mM Tris HCl (pH~8.3)
* 0.1 mg/mL MgCl2, 6H20
* 0.1 mg/mL Gelatin
* 0.45% (NP-40) Jgepal
* 0.45% Tween 20

==Protocol==
# Combine 100-200uL of PBND solution with 2ul of Proteinase K and mouse tail in an eppindorf tube or in a well of a 96 well PCR plage (Proteinase K stock = 10mg/mL)
# Incubate at 55 degrees (O/N)
# Incubate at 85 degrees for 45 min
# Run PCR

[[Category: Mouse Work]]
[[Category: Molecular Biology]]
[[Category: DNA]]
[[Category: Genotyping]]

Triglyceride Assay from Cells and Tissues

2013-06-19T15:29:31Z

Mpeloqu1: Deletion of unnecessary information (1/10 Glycerol Dilution)

==Materials==
* '''Homogenization Buffer''' (50 mM Tris pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
* 10M KOH
* '''Chloroform/Methanol Mixture''' (2:1)
* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
* Sigma Triglyceride Assay Kit (Cat TR0100)

==Protocol==
# Weigh out 30 mg tissue (record weight for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
# Add 500 uL Homogenization Buffer.
# Homogenize with qiagen tissue lyser 3 minutes at 25 Hz for Liver/WAT or 5 min at 30 Hz for muscle.
# Add 12.5 uL KOH
# Mix by inverting
# Add 800 uL '''Chloroform/Methanol Mixture'''
# Vortex vigorously then sit at room temperature for 5 min
# Centrifuge 10min at 13 000 RPM
# Take the bottom layer into a fresh labelled tube.
# Dry in fume hood overnight (or until completely dry)
# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
# Add 50uL '''(500uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
## Resuspend triglyceride and glycerol reagent with water if necessary.
## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
## Prepare reagent, you need 560 uL '''(80uL)''' of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent. Make a bit extra and combine in a falcon tube.
## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
## For standards add 0-5 uL of glycerol standard.
## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
## Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry). If using > 10 uL of butanol mixture the solution may be cloudy, let it settle and it should become more clear.
## Measure absorbance at 540 nm.
## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.

Triglyceride Assay from Cells and Tissues

2013-06-19T15:24:20Z

Mpeloqu1: updated volumes and lysis procedure

==Materials==
* '''Homogenization Buffer''' (50 mM Tris pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
* 10M KOH
* '''Chloroform/Methanol Mixture''' (2:1)
* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
* Sigma Triglyceride Assay Kit (Cat TR0100)

==Protocol==
# Weigh out 30 mg tissue (record weight for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
# Add 500 uL Homogenization Buffer.
# Homogenize with qiagen tissue lyser 3 minutes at 25 Hz for Liver/WAT or 5 min at 30 Hz for muscle.
# Add 12.5 uL KOH
# Mix by inverting
# Add 800 uL '''Chloroform/Methanol Mixture'''
# Vortex vigorously then sit at room temperature for 5 min
# Centrifuge 10min at 13 000 RPM
# Take the bottom layer into a fresh labelled tube.
# Dry in fume hood overnight (or until completely dry)
# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
# Add 50uL '''(500uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
## Resuspend triglyceride and glycerol reagent with water if necessary.
## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
## Prepare reagent, you need 560 uL '''(80uL)''' of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent. Make a bit extra and combine in a falcon tube.
## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
## For standards add 0-5 uL of glycerol standard '''(or of a 1/10 dilution of the glycerol standard)'''.
## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
## Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry). If using > 10 uL of butanol mixture the solution may be cloudy, let it settle and it should become more clear.
## Measure absorbance at 540 nm.
## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.